Article excerpt

A growing number of substances released into the environment disrupt normal endocrine mechanisms in a wide range of vertebrates. Little is known about the effects and identities of endocrine-disrupting chemicals (EDCs) that target thyroid hormone (TH) action, particularly at the cellular level. Frog tadpole metamorphosis depends completely on TH, which has led to the suggestion of a metamorphosis-based assay for screening potential EDCs. A major mechanism of TH action is the alteration of gene expression via hormone-bound nuclear receptors. To assess the gene expression profiles in the frog model, we designed a novel multispecies frog cDNA microarray. Recently, the preemergent herbicide acetochlor was shown to accelerate 3,5,3'-triiodothyronine ([T.sub.3])induced forelimb emergence and increase mRNA expression of thyroid hormone [beta] receptors in ranid tadpoles. Here we show that [T.sub.3]-induced metamorphosis of Xenopus laevis a species commonly used in the laboratory, is accelerated upon acute exposure to an environmentally relevant level of acetochlor. The morphologic changes observed are preceded by alterations in gene expression profiles detected in the tadpole tail, and the nature of these profiles suggest a novel mechanism of action for acetochlor. Key words: acetochlor, cDNA array, endocrine disruptor, gene expression, herbicides, metamorphosis, persistent organic pollutants, tadpoles, thyroid hormone, Xenopus laevis. Environ Health Perspect 110:1199-1205 (2002). [Online 7 October 2002]

Anuran metamorphosis is a rapid, complex postembryonic process in which the tadpole transforms into a juvenile frog. The dramatic structural and functional changes of larval tissues during metamorphosis depend completely on an elevation of endogenous thyroid hormones (TH), 3,5,3'-triiodothyronine ([T.sub.3]), and 3,5,3',5'-tetraiodothyronine ([T.sub.4]) (Gilbert et al. 1996; Shi 2000). The major mechanism of TH action involves hormone binding to nuclear TH receptors (TR[alpha] and TR[beta]), resulting in tissue-specific activation/repression of gene transcription (Helbing and Atkinson 1994; Helbing et al. 1992, 1996; Sachs et al. 2000; Shi 2000; Tata et al. 1993). Studies using subtractive hybridization have identified components of these genetic programs initiated during amphibian metamorphosis. In Xenopus laevis, TH differentially affects the expression of 45 genes in the tail, 34 genes in the brain, and more than 120 genes in the limb within the first 48 hr of precocious metamorphosis (Brown et al. 1996; Buckbinder and Brown 1992; Denver et al. 1997; Wang and Brown 1993). With the development of DNA microarray technology, high throughput analysis of the expression profiles of hundreds to thousands of genes altered during metamorphosis is possible. The absolute requirement for TH action has led to the consideration of amphibian metamorphosis as a standardized method for screening potential endocrine-disrupting chemicals (EDCs), many of which are environmental contaminants (Crump 2001; De Vito et al. 1999; Hutchinson et al. 2000). Indeed, DNA microarrays have been used to detect genotoxic effects after exposure to chemical contaminants (Bartosiewicz et al. 2001; Custodia et al. 2001; Lobenhofer et al. 2001; Nuwaysir et al. 1999).

The preemergent herbicide acetochlor [2-chloro-N-(ethoxy-methyl)-N-(2-ethyl-6-methylphenyl) acetamide] is a persistent organic pollutant (POP) that can be detected in shallow ground water 1 year after field application, and there is evidence that it can act as an EDC (Ashby et al. 1996; Barbash et al. 1999; Cheek et al. 1999a; Veldhoen and Helbing 2001; Wilson et al. 1996). Acetochlor was introduced in 1994, and its use in agriculture has increased approximately 32% measured as the number of pounds applied annually (from 1994 to 2000). …